Botulinum NeuroToxin (BoNT) is a 150 kDa protein produced by anaerobic bacteria species, most notably Clostridium botulinum, that causes life-threatening botulism. BoNT causes disease by preventing the release of neurotransmitters at distinct synapses (Humeau, Doussau et al. 2000), blocking nerve impulses and resulting in a flaccid paralysis. Release of neurotransmitters is blocked by proteolytic activity of a 50 kDa fragment of BoNT called the light chain (LC). This is a Zn2+ containing endoprotease.
There are seven known serotypes of BoNT, labelled A to G, which are distinguished from each other by antibody neutralization assays. Type A is commonly associated with botulism in humans. Crystalline BoNT-A (Mw=900,000) consists of two BoNT Type A molecules (Mw=150,000) and a number of non-toxic Neurotoxin Associated Proteins (NAP) that help shield the toxin against acidic and proteolytic attack in the gastro intestinal tract.
Detection of BoNT at relevant concentrations is challenging because it is highly lethal and therefore a test for it must be correspondingly sensitive. Lethal amounts of crystalline BoNT type A per kg body weight in humans is estimated from primate studies to be 1 μg when taken orally, 1.3-2.2 ng intravenously or intramuscularly, and 10-12.9 ng by inhalation. Assuming 70 kg body weight and 5 liters of peripheral blood for an average adult, this would correspond to a lethal concentration of 18.2-30.8 ng/L crystalline BoNT/A (0.040-0.069 pM BoNT/A).
The currently accepted test for BoNT is the standard mouse bioassay. This requires intraperitoneal injection of two or more 20-30 g mice with 0.4-0.5 ml of filter sterilized sample, and watching for signs of intoxication. BoNT intoxicated mice will usually die within 6-96 hours, depending on the level of toxin in the sample. The LD50 of crystalline BoNT Type A for mice is 0.03 ng which corresponds to 5 pg of pure BoNT-A (Schantz and Johnson 1992).
The mouse bioassay causes severe distress, cannot be used in the field, is time consuming, cost intensive and it is impractical for screening large numbers of samples. In response to the need for a rapid and sensitive assay for BoNT, in vitro assays have been under development in recent years.
Assays that detect BoNT proteolytic activity employ naturally occurring or synthetic substrates of LC, together with methods for detecting the cleaved products. These include antibodies against the cleaved fragments, mass spectrometry, and fluorescence.
The first fluorescence-reporter assays employed fluorescein markers immobilized by cleavable peptides that were released by BoNT proteolytic activity, or Förster Resonance Energy Transfer (FRET) between a chromophore quencher and a fluorophore. FRET was inactivated by BoNT proteolytic activity and this was detected by changes in fluorescence. Other fluorescence-based assays for proteolytic activity were subsequently developed using similar principles of operation: either detection of released fluorophores, or fluorescence signals associated with FRET.
WO 2004/031355 A2 describes an assay for the detection of botulinum toxin based on proteolytic cleavage of SNAP25. The construct used comprises a hexahistidine tag and a fluorescent molecule for detection. The sensitivity of the assay (FIG. 3B) is said to be down to 1.0 ng/ml BoNT/A holotoxin (6.6 pM). A higher sensitivity of an assay for the detection of botulinum toxin is desirable (<0.1 pM).